The poor response of existing treatnnent options for unresectable pancreatic cancer is the result of a subset of cancer cells exhibiting chemoresistance. Evidence has shown that the capacity of a tumor to grow and propagate is dependent on this small subset of cells, identified as pancreatic cancer stem (or stem-like) cells (CSCs). Due to their vital role in tumor maintenance and the formation of metastases, CSCs are integral targets to treat pancreatic cancer, as current therapeutic regimens cannot effectively treat this cell population because of its intrinsically resistant nature. Adenovirus (Ad)-based oncolytic viruses have unique advantages for targeting and eradicating pancreatic CSCs along with inclusively targeting the general pancreatic cancer cell population and thereby represent a promising novel therapeutic strategy. Ad replication specificity can be achieved by placing viral replication genes under the control of tumor-specific promoters, thereby generating conditionally replicative adenoviruses (CRAds). The keys to developing a pancreatic cancer CRAd that inclusively targets CSCs are: 1) effective transduction and 2) replication specificity of pancreatic cancer cells and CSCs. We have developed innovative strategies and breakthrough technologies which can be rapidly applied for designing such CRAds. Our advanced infectivity enhancement strategies allow efficient transduction of pancreatic cancer cells and CSCs. To confer replication specificity, we have developed the CXCR4 promoter as a CRAd replication control element. CXCR4 expression Is a signature of pancreatic cancer and CSCs. SA 1: To construct infectivity-enhanced, CXCR4 promoter controlled CRAds for pancreatic cancer, inclusively targeting pancreatic CSCs. SA 2: To validate the efficacy of CRAds with in vitro models of chemoresistant pancreatic CSCs. SA 3: To demonstrate efficacious targeting and replication of CXCR4 CRAds with in vivo models of pancreatic cancer. SA 4: To validate the efficacy and specificity of pancreatic CSC-targeted CRAds with primary CSCs derived from human patient tumors. Development of an effective therapy for the advanced pancreatic cancer Is predicated on efficiently targeting and eradicating pancreatic CSCs, which represent the chemoresistant nature of this disease. The strategy proposed herein, of exploiting CRAd agents based on the CXCR4 axis to inclusively target pancreatic CSCs offers a promising treatment approach. On the basis of these studies, we will thus understand the direct utility of our novel system for clinical application and will facilitate a rapid clinical translation. We will submit an IND application concurrently with this proposal to rapidly translate the agents herein for clinical trial.